The extreme remodeling of pods by St and To eliminates pod shattering, but makes pods extremely difficult to thresh , and the alleles are therefore impractical for dry bean market classes. Since arid conditions are predicted to increase in coming decades , shattering-resistance alleles will be of increasing value for plant breeders. Despite this, little information exists on the degree of pod shattering in major market classes, the pattern of dominance and epistasis between resistance alleles, or the diversity available at each of these loci. Crucially, breeders also still lack genetic assays to evaluate the trait in segregating populations. Addressing these barriers will be critical to improve the productivity of a major source of nutrition globally. Three populations were evaluated in this study: a biparental population and two diversity panels, which represent each of the two domestication events of common bean. The biparental population was developed to study two shattering-related QTLs, their patterns of dominance and their interactions. Cultivars ‘Maylower’ and ‘Bill Z’ showed total resistance to pod shattering when field grown in Davis in 2017 . These varieties were among the most distantly related accessions in the MDP, with neither showing any evidence of admixturebetween ecogeographic races . May lower is a navy bean type , which possesses a SNP allele on Pv08 that is weakly associated with resistance to pod shattering in race Mesoamerica. Bill Z is a pinto bean type and has a SNP variant on Pv03 associated with strong PvPdh1-mediated shattering resistance common in race Durango. The population can therefore be used to determine if a reduction in pod shattering was independently selected in each of these ecogeographic races. An F3 population of 138 individuals was developed by hybridization between these cultivars. Each F3 individual was descended from a distinct F2 plant,best vertical garden system and all of the F2 s were the progeny of a single F1 developed by cross-pollinating Maylower and Bill Z. This 138-member Maylower x Bill Z population was used to validate the possible alleles on Pv03 and Pv08 and test any patterns of dominance and epistasis between the loci.
The two diversity panels were grown to evaluate the degree of pod shattering across diverse accessions of common bean. In 2016, 98 members of major market classes in the Andean Diversity Panel were ffield-grown in Davis, California, to evaluate each variety’s susceptibility to pod shattering. In 2017, 278 varieties of the BeanCAP Middle American Diversity Panel were similarly ffield-grown in Davis to evaluate pod shattering. At maturity, a sample of pods was harvested from each variety. Mature pods of all phenotyped varieties were harvested and then exposed to seven days of desiccation at 65 °C and a further seven days of re-equilibration to room temperature. The desiccation conditions for all varieties were identical, and desiccation was conducted using the same drying chamber. The proportion of pods dehiscing in this treatment was recorded, along with the market class of each variety. For evaluation of pod twists in the MxB population, all non-shattering pods were fractured by hand, and then, all pods were subjected to the desiccation treatment and re-equilibration again. The number of twists was counted for ten pods of each genotype, with “1” indicating a complete 360° rotation of the valve.DNA was extracted from young trifoliate leaves of the greenhouse-grown biparental MxB F3 generation, using a modiied CTAB protocol . DNA was quantiied with a NanoDrop spectrophotometer and genotyped using the BARCBean6K_3 BeadChip , yielding 5398 initial SNPs. SNPs that were missing or heterozygous in either parent or identical between the parents, were filtered from further analysis. The remaining SNPs were arranged into a linkage map using the ASMap R package . SNPs that did not map to one of the 11 major linkage groups were removed, leaving 1794 SNPs for QTL mapping. QTL mapping was conducted using the expectation maximization method in R/qtl . Phenotypes for QTL mapping were generated by harvesting all the pods from each greenhouse-grown F3 plant , then subjecting them to seven days at 65 °C and seven further days of re-equilibration to room temperature. Pods that had fractured to the tip of the beak due to this treatment were counted as shattered, while those with no opening or only issuring along the sutures were considered non-shattering.The maximum LOD score of 1000 randomized analyses of the data was used as a significance threshold. To test dominance, F3 individuals were subset by genotype at highly significant SNPs, and comparisons were made between groups by t-test.Next, the 43 SNPs within 100 kb of PvPdh1 in the MDP data set were analyzed to identify patterns of selection and diversity around the gene.
To simplify and visualize the data, principal component analysis was performed on the SNPs using R. Sequence variation was converted to integer values and the imputePCA function of the missMDA package was used to impute missing data . The genotype data were also sorted to identify unique haplotypes within the populations. The degree of similarity between the PCA and haplotype diversity was then compared. Individuals with missing data for SNPs distinguishing the haplotypes or haplotype clusters were not shown in plots and not numbered in plots as they could not be unambiguously placed within any haplotype group.The PvPdh1 putative causal polymorphism was used to develop a Cleaved Ampliied Polymorphic Sequence marker for efficient screening of breeding populations. The sequence surrounding the SNP was extracted using Phytozome 12 . Restriction enzymes that would differentially cut the alternative alleles were identified using RestrictionMapper version 3.0 . PCR primers were developed for the locus based on the sequence of accession G19833 , using the NCBI primer BLAST tool, and were then checked against the genome sequence of BAT93 to ensure that the sequences were identical and would successfully amplify membersof both major gene pools. The sequence surrounding the SNP was ampliied using the primers PDH1-TAQII-2F and PDH1-TAQII-2R . PCR was conducted with Takara ExTaq and included an initial elongation at 95 °C for three minutes, 44 cycles with denaturation at 95 °C for 30 s, annealing at 54 °C for 30 s, elongation at 72 °C for 60 s, and a final elongation of 72° for five minutes. PCR products were cleaved with ChimerX TaqII during a 65 °C degree incubation for seven hours, and run on a 2.5% agarose gel. The SNPs tightly linked to PvPdh1 in the MDP data set were then screened for other positions that could be useful for conversion to additional CAPS markers. The SNP closest to PvPdh1 in this data set, at Pv03 position 49,132,438 , is distinguishable by EcoRI and is highly correlated with pod shattering. Unlike the TaqII-based CAPS marker, the allele cleaved by EcoRI is the shattering-resistant variant, reducing the risk of falsely identifying a susceptible individual as resistant due to technical errors in digestion. The SNP distinguished by EcoRI is separated from the PvPdh1 causal polymorphism by less than 7 kb. The sequence surrounding this SNP was ampliied using the primers PDH1-ECORI-1F and PDH1-ECORI-1R . Marker development used the same methods as the TaqII-distinguishable marker, and the same PCR conditions successfully amplified both fragments.
The amplicons were then digested by Promega EcoRI at 37 °C for 15 min, and the PCR products were resolved on a 2.5% agarose gel. Major discrepancies in pod shattering exist between the major market classes of common bean . In the Andean gene pool, pod shattering is highest in the cranberry market class,vertical farming equipment with a mean value of 41% of pods shattering after desiccation. The purple speck/mottled market class has the greatest degree of shattering resistance among Andean beans, with only 3% of pods shattering after the same treatment. In Middle American beans, pod shattering is highest in the black and navy/small white market classes of race Mesoamerica, and lowest in the pinto , great northern and pink classes of race Durango. PvPdh1-mediated resistance to pod shattering is found almost exclusively in pinto, great northern, and pink market classes and is therefore associated with levels of pod shattering which may be the lowest of those of any major economic groups of common beans grown for grain.Three major SNP haplotype clusters were identified in the sequence surrounding the PvPdh1 gene . The most distinct of these included six individuals, several of which are of known Andean ancestry. The first principal component of the genetic data explained 64% of the variation and separated this group from the two other major clusters. The second principal component explained 25% of the variation and separated varieties belonging to race Mesoamerica from race Durango. Five individuals with missing data for the ten SNPs that distinguish these ecogeographic races were filtered from subsequent analyses. Additionally, cv. ‘Tepary 22′ and cv. ‘Jackpot’ exhibited highly unique haplotype patterns. Jackpot shows recombination in the region between the predominant haplotype of the Andean gene pool and race Durango, while P. acutifolius is a separate but closely related species . Races Durango and Mesoamerica differed in haplotype diversity around the PvPdh1 locus . The race Mesoamerica haplotype cluster includes six unique haplotypes. The most common of these includes 137 of the 148 varieties that can be clustered into a group unambiguously , without missing data in the SNP positions distinguishing the sub-groups. The race Mesoamerica haplotypes displayed 18% shattering on average. In contrast, race Durango varieties display only three haplotypes. The most common of these haplotypes includes 178 of 182 unambiguous varieties , with an average proportion shattering of 0.6% in this group. The two low-frequency race Durango haplotypes showed no shattering when field-grown in 2017 .The TaqII-based CAPS marker of the PvPdh1 causal polymorphism leads to cleavage of susceptible alleles, while resistant alleles are not cut. The total pre-digestion amplicon length in G19833 was 578 bp, comparable to the 580 bp amplicon of BAT93. After digestion, susceptible alleles were cleaved into fragments of 449 and 129 bp in G19833. While digestion was seen in all shattering-susceptible samples after digestion with TaqII, this enzyme led to only partial digestion in a minority of cases. The EcoRI-digestible CAPS marker was extremely robust, and never led to partial digestion. After digestion, this marker led to resistant alleles that were cut into fragments of approximately 332 bp and 310 bp, while susceptible alleles remained uncleaved at 642 bp in BAT 93 . Andean varieties showed comparable fragment sizes, such as 639 bp in G19833. The EcoRI-digestible CAPS marker never experienced partial digestion or ambiguity. The SNP used for this marker has a strongly significant correlation with pod shattering , and is one of the 10 SNPs contributing to the haplogroup differentiation between race Durango and race Mesoamerica . The median proportion of pod shattering among 97 varieties with the shattering susceptible allele was 0.14, equal to the maximum level of shattering seen in any of the 160 varieties carrying the resistant allele . Themedian proportion shattering of those varieties with the resistant allele was 0.00%. Testing for multiple origins of Middle American shattering resistance and allelic effects Only one major locus was identified with an effect on pod shattering in the Middle American domesticated gene pool of common bean. These results highlight the important role of the Pv03 PvPdh1 locus in this population. The lack of a major QTL on Pv08 in the MxB population suggests that Maylower has no shattering resistance allele on that chromosome which is not also found in the distantly related Bill Z. Because the Pv08 SNP identified through GWAS did not reach significance after a Bonferroni correction and only 11 of 280 members of the MDP possessed this SNP, our results indicate that this chromosome does not have a major, widespread role in regulating pod shattering in the Middle American domesticated gene pool. This does not preclude the possibility that the QTL has a role in shattering of Andean beans, the latter of which has been demonstrated with much greater conidence . The locus may also have a role in regulating pod shattering in a very small proportion of Middle American bean varieties, possibly through de novo mutation in race Mesoamerica or introgression from the Andean gene pool.